Figure 5.
Translation of C/EBPβ isoforms is altered under stress conditions. (A) Structures of the 3 C/EBPβ isoforms. The regions recognized by the 2 antibodies are indicated. (B) Immunoblot analysis of NIH3T3 cell extracts with the anti-N terminus (anti-N) and anti-C terminus (anti-C) antibodies. The anti-N antibody recognized only LAP* and LAP, whereas the anti-C antibody detected all 3 isoforms. (C) Flow cytometric analysis of EML cells transduced with control, LIP, LAP, or LAP* expression vector. Cells were not selected after transduction, so the results reflect a mixture of transduced and nontransduced cells. Blue lines indicate the expression level in cells transduced with control vector. (D) Intracellular double staining of LT-HSCs (CD150+CD48− KSL cells) obtained from 5-FU–treated WT mice with the 2 anti-C/EBPβ antibodies. Representative flow cytometric patterns at multiple time points after 5-FU administration are shown. Blue lines indicate the expression level in LT-HSCs at steady state stained under the same conditions. (E) Changes in total C/EBPβ protein (MFIC/EBPβ C-term) and (F) C/N ratio (ratio of MFIC/EBPβ C-term/MFIC/EBPβN-term) in LT-HSCs at multiple time points after 5-FU administration. The C/N ratio at steady state was set to 1 (dashed line). The effect of rapamycin administration (from day −7) was also compared (green line) with that of vehicle administration (black line) (n = 4–6 per group and per time point in 2 independent experiments). (G) LIP is specifically upregulated in CD150high myeloid-biased LT-HSCs (CD150+CD48− KSL cells) after 5-FU treatment. Total C/EBPβ protein level in CD150high and CD150low LT-HSCs 3 days after 5-FU administration. (i-ii) Representative histograms of fluorescence corresponding to anti-C antibody staining in CD150high and CD150low LT-HSCs (red and black lines, respectively) are shown. (iii-iv) Surface CD150 expression was assessed separately in C/EBPβ(C-term)high and C/EBPβ(C-term)low LT-HSCs (blue and black lines, respectively) 3 days after 5-FU administration. (H-I) Total C/EBPβ protein level (MFIC/EBPβ C-term) and the C/N ratio (ratio of MFIC/EBPβ C-term/MFIC/EBPβN-term) in CD150high (red line) and CD150low (black line) LT-HSCs (CD150+CD48− KSL cells) at multiple time points after 5-FU administration (n = 4-6 per group and per time point in 2 independent experiments). The C/N ratio at steady state was set to 1 (dotted line). Data are presented as means ± SD. *P < .05; **P < .01; and ***P < .001 (determined by the 2-tailed Student t test). BR-LZ, basic region-leucine zipper domain; RD, regulatory domain; TAD, transactivation domain.

Translation of C/EBPβ isoforms is altered under stress conditions. (A) Structures of the 3 C/EBPβ isoforms. The regions recognized by the 2 antibodies are indicated. (B) Immunoblot analysis of NIH3T3 cell extracts with the anti-N terminus (anti-N) and anti-C terminus (anti-C) antibodies. The anti-N antibody recognized only LAP* and LAP, whereas the anti-C antibody detected all 3 isoforms. (C) Flow cytometric analysis of EML cells transduced with control, LIP, LAP, or LAP* expression vector. Cells were not selected after transduction, so the results reflect a mixture of transduced and nontransduced cells. Blue lines indicate the expression level in cells transduced with control vector. (D) Intracellular double staining of LT-HSCs (CD150+CD48− KSL cells) obtained from 5-FU–treated WT mice with the 2 anti-C/EBPβ antibodies. Representative flow cytometric patterns at multiple time points after 5-FU administration are shown. Blue lines indicate the expression level in LT-HSCs at steady state stained under the same conditions. (E) Changes in total C/EBPβ protein (MFIC/EBPβ C-term) and (F) C/N ratio (ratio of MFIC/EBPβ C-term/MFIC/EBPβN-term) in LT-HSCs at multiple time points after 5-FU administration. The C/N ratio at steady state was set to 1 (dashed line). The effect of rapamycin administration (from day −7) was also compared (green line) with that of vehicle administration (black line) (n = 4–6 per group and per time point in 2 independent experiments). (G) LIP is specifically upregulated in CD150high myeloid-biased LT-HSCs (CD150+CD48− KSL cells) after 5-FU treatment. Total C/EBPβ protein level in CD150high and CD150low LT-HSCs 3 days after 5-FU administration. (i-ii) Representative histograms of fluorescence corresponding to anti-C antibody staining in CD150high and CD150low LT-HSCs (red and black lines, respectively) are shown. (iii-iv) Surface CD150 expression was assessed separately in C/EBPβ(C-term)high and C/EBPβ(C-term)low LT-HSCs (blue and black lines, respectively) 3 days after 5-FU administration. (H-I) Total C/EBPβ protein level (MFIC/EBPβ C-term) and the C/N ratio (ratio of MFIC/EBPβ C-term/MFIC/EBPβN-term) in CD150high (red line) and CD150low (black line) LT-HSCs (CD150+CD48− KSL cells) at multiple time points after 5-FU administration (n = 4-6 per group and per time point in 2 independent experiments). The C/N ratio at steady state was set to 1 (dotted line). Data are presented as means ± SD. *P < .05; **P < .01; and ***P < .001 (determined by the 2-tailed Student t test). BR-LZ, basic region-leucine zipper domain; RD, regulatory domain; TAD, transactivation domain.

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